Treatment of ocular wounds and ulcers

ABSTRACT

A method for treating ocular conditions such as bacterial keratitis, bacterial conjunctivitis, corneal ulcers and wounds, endophthalmitis, and blebitis in mammals, by using a native, synthetic, or recombinant CAP37, or effective peptide portions thereof including CAP37 peptides 20-44, 23-42, 102-122, and 120-146 and monocysteine derivatives of peptides 20-44 and 23-42. The CAP37, peptides, and peptide derivatives can also be used to store, clean, sterilize, or coat contact lenses, and may be used in media for storing mammalian corneal transplants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/423,311, filedApr. 25, 2003, which claims the benefit of U.S. Ser. No. 60/378,295,filed May 3, 2002, each of which is hereby expressly incorporated hereinby reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Some aspects of this invention were made in the course of Grant AI 28018awarded by the National Institutes of Health and therefore theGovernment has certain rights in some aspects of this invention.

BACKGROUND

Ocular infections such as bacterial keratitis are serious clinicalproblems. Bacterial keratitis, for example, is a component of manyocular infections, especially among those who have sustained penetratingcorneal injuries, used extended-wear contact lenses, undergoneincisional refractive surgery, or are immunocompromised. Bacterialkeratitis is an important cause of visual morbidity. Contact lenswearers are most at risk. More recently, the use of refractivecorrection in the form of incisional and laser surgery has emerged as anew cause of bacterial keratitis (1-4). Loss of vision and permanentscarring are commonly due to toxic bacterial products and the hostinflammatory response to wounding and infection. Common causativeorganisms are the Gram positive bacteria Staphylococcus aureus and theGram negative bacterium Pseudomonas aeruginosa (5-7). The bacterialproducts and toxins and host inflammatory reaction stimulated inresponse to wounding and infection often leads to extensive tissuedamage with permanent scarring and irreversible loss of vision (1).

Current treatments include the use of broad spectrum antibiotics.Topical antibiotic drops are the preferred treatment for corneal andconjunctival infections. Intravitreal antibiotics are preferred forendophthalmitis and parenteral antibiotics are recommended for deepinfections.

The diagnosis and treatment of bacterial keratitis remainscontroversial. A combination of a fortified topical cephalosporin and afortified topical aminoglycoside were once the first line of therapy.However, recently this therapy has been replaced by fluoroquinolonessuch as ciprofloxacin and oflaxacin for topical ophthalmic therapy.However, the emergence of methicillin-resistant organisms has reducedthe effectiveness of these antibiotics. Thus the choice of initialempirical therapy is controversial. Clearly, there is a crisis situationdeveloping with organisms that cause ocular infections which areresistant to antibiotics.

Because early treatment of the infection is important to prevent loss ofvision, treatment is generally started before the specific identity ofthe causative organism and its sensitivity are known. Therefore, a broadspectrum antibiotic is generally used initially. Once the cultureresults are known the treatment is best modified to a single drug tocover the infectious organisms. It is important that the specificantibiotic have as narrow a spectrum as possible, since broad spectrumagents could unnecessarily alter the normal flora allowing superinfection from resistant or nonsusceptible organisms.

Steroid treatment has also been used in conjunction with antibiotics inthe hope that it will limit the inflammatory process of the host,however this course of treatment requires careful monitoring.

Almost all topical ophthalmic antibiotics can cause local irritation andallergic reactions. Treatment for severe bacterial keratitis (bacterialcorneal ulcer), regardless of the identity of the antimicrobial agentused, typically consists of instillation of drug every 15-30 minutesaround the clock for the first 2-3 days. The dosing interval is thengradually increased to every four hours and continued for an additional14 days. Topical drops are preferred for corneal and conjunctivalinfections. The agent should be bactericidal rather than bacteriostatic.

The cornea is normally considered a “privileged” site because of itsavascularity and lack of lymphatic vessels (8-10). Antigens, cytokines,inflammatory mediators and leukocytes that enter into the cornea must doso from the limbic and/or ciliary body vessels. Inflammatory cytokinesand/or chemotactic gradients that are elicited locally by corneal cellscould therefore profoundly affect the emigration of leukocytes from thelimbic and ciliary circulation to the cornea.

Extravasation of leukocytes from the circulation into tissue sites is anintegral feature of the host response to injury and inflammation. Byvirtue of their ability to engulf and destroy bacteria, eliminate toxinsand secrete numerous soluble mediators, leukocytes are capable ofrestricting and limiting the spread of infection. Neutrophils (PMNs) arethe predominant cell type in the early phases of inflammation and aresoon followed by a second wave of cells composed mainly of monocytes andlymphocytes. Irreversible damage to the eye can occur in cases offulminant inflammation. Clearly the desirable outcome is one in whichthe immune system can control the infection resulting inre-epithelialization and healing with minimum damage to vision.

The identification of a corneal derived chemotaxin or inflammatorymediator could be of extreme importance in our understanding of themechanisms that regulate leukocyte migration, epithelial-leukocyteinteraction, corneal inflammation and healing and in identifying methodsof treatment of corneal damage related to infection, inflammation andphysical wounding.

SUMMARY OF THE INVENTION

Pseudomonas aeruginosa is frequently associated with infection followinguse of extended-wear contact lenses. The most common organism associatedwith corneal infection in patients who do not wear contact lenses isStaphylococcus aureus. CAP37 is important in the recruitment ofleukocytes from the circulation in the limbus of the eye to theavascular cornea. CAP37 proteins and peptides derived therefrom can beused as a topical/oral/intravenous/intravitreal antibiotic for thetreatment of ocular bacterial infections in mammals including humans,primates, rabbits, livestock animals and ungulates, for example. CAP37and CAP37 peptides can also be used to promote healing of corneal woundsand ulcers that may not have an infective component, such as those dueto injury by foreign objects or trauma. CAP37 and CAP37 peptides canalso be used to treat contact lenses, to sterilize the lenses andinhibit infections caused by bacteria on the lenses. Mammalian cornealtransplants can also be stored in media containing CAP37 and/or CAP37peptides as described herein.

Corneal wound healing consists of three interrelated processes,including corneal epithelial cell proliferation, corneal epithelial cellmigration and upregulation of adhesion molecules that are capable ofbinding to extracellular matrix proteins forming attachments andadhesion and thereby aiding healing. As shown herein, CAP37 promotescorneal epithelial cell proliferation, and migration. Also shown is thatCAP37 upregulates corneal epithelial cell adhesion molecules includingintercellular adhesion molecule-1 (ICAM-1) and platelet-endothelial celladhesion molecule-1 (PECAM-1). Both ICAM-1 and PECAM-1 are important inleukocyte-epithelial interactions. Importantly CAP37 upregulates α-3(CD49c) and β-1 (CD29) integrin molecules. α-3 β-1 integrin moleculesare critical for binding of the corneal epithelial cell to laminin-5 andfibronectin two important constituents in the basement membrane of thecornea. Taken together these studies indicate that CAP37 is involved inthe promotion of corneal epithelial wound healing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Ocular localization of CAP37 in response to intrastromalinjection of S. aureus in the rabbit eye-model of bacterial keratitis.(a) A representative photomicrograph of the junction between ocularconjunctiva and cornea at 5 hr post infection. Immunohistochemicalstaining using mouse anti-CAP37 and the Vectastain ABC-peroxidasetechnique indicating faint staining for CAP37 in the conjunctivalepithelium and relatively weaker staining for CAP37 in cornealepithelium. Note absence of staining in vascular endothelium, ×400 (b)Sham-injected rabbit eye at 5 hr stained with mouse anti-CAP37 antiserumindicating absence of staining for CAP37 in ocular tissue, ×400 (c)Immunohistochemical staining for CAP37 using mouse anti-CAP37 antiserumat 10 hr post infection showing strong reaction for CAP37 in cornealepithelium, ×400 (d) Strong staining for CAP37 in endothelial cellslining a vessel located in the ciliary body at 10 hr post infection.Also note staining for PMN, ×1000 (e) Specificity control using normalmouse serum to stain tissue from a rabbit 10 hr post infection, ×400 (f)Absence of staining with immunoadsorbed anti-CAP37 antiserum in cornealepithelium obtained from rabbit 10 hr post infection, ×100 (g)Immunohistochemical localization of CAP37 in rabbit eye 15 hr postinfection. Strong positive reaction for CAP37 is observed in cornealepithelium as well as in infiltrating PMN in corneal stroma and stromalkeratocytes, ×400 (h) Immunohistochemical localization of CAP37 20 hrpost infection indicating reduced levels of staining for CAP37 incorneal epithelium. Note continued strong staining for CAP37 in PMN atthe base of the epithelial layer, ×400.

FIG. 2. In vitro induction of CAP37 in human corneal epithelial cells(HCEC) and stromal keratocytes. (a) Representative figure indicatingimmunohistochemical detection of CAP37 protein using mouse anti-CAP37antiserum and the Vectastain ABC-peroxidase staining technique on HCECtreated with tumor necrosis factor-α (TNF-α 5 ng/ml) for 24 hr, ×200 (b)Induction of CAP37 in HCEC in response to Interleukin-1β (IL-1β 10ng/ml) treatment for 24 hr, ×400 (c) Staining using mouse anti-CAP37antiserum on untreated HCEC indicating absence of staining for CAP37,×100 (d) Antibody control using immunoadsorbed anti-CAP37 antiserum onHCEC treated with IL-1β (10 ng/ml) for 24 hr, ×200 (e) Induction ofCAP37 in stromal keratocytes in response to TNF-α (10 ng/ml for 24 hr)as detected immunohistochemically using mouse-anti-CAP37 antiserum, ×400(f) Immunoadsorbed anti-CAP37 antiserum control indicating absence ofCAP37 in stromal keratocytes treated with TNF-α (10 ng/ml for 24 hr),×400.

FIG. 3. Kinetic study demonstrating the effect of proinflammatorycytokines on steady state levels of CAP37 mRNA in corneal epithelialcells. (a) HCEC were treated (+) with TNF-α (5 ng/ml) for 5 min, 15 min,30 min, 2 hr, 4 hr and 6 hr and CAP37 mRNA expression (upper panel, 597bp) determined by RT-PCR. Untreated (−) HCEC controls are included foreach time point. Lane 13 is a negative-water control. Lane 14 is thepositive HL-60 control. (b) HCEC were treated with IL-1β (10 ng/ml) for0.5, 1, 4, 6, and 8 hr and CAP37 mRNA expression (upper panel 597 bp)determined by RT-PCR. Untreated (−) controls are included for eachincubation point. Lane 1 is the positive HL-60 control and lane 2, thenegative-water control. The lower panel in both indicates cDNA integrityas assessed with the β-actin primer (267 bp). Molecular markers arepresent in unmarked lanes in both panels.

FIG. 4. Upregulation of intercellular adhesion molecule-1 (ICAM-1) onhuman corneal epithelial cells in response to CAP37. (a) Dose responseeffect of CAP37 on expression of ICAM-1 on cultured HCEC. Cells weretreated with CAP37 (0-2000 ng/ml) for 6 hr and stained with FITC-labeledmouse anti-human ICAM-1 and fluorescence intensity measured by flowcytometry. Controls included the isotype IgG₁ antibody and TNFα (5ng/ml), which served as the positive control. Values are mean±SE ofresults obtained from 9 independent experiments. **P<0.01 compared tountreated control. (b) Kinetic response of CAP37-mediated expression ofICAM-1 in HCEC. Cells were treated with 1000 ng/ml of CAP37 (circles) at2, 6, 24, 48, & 72 hr and ICAM-1 expression analyzed by flow cytometry.TNF-α at 5 ng/ml (squares) was used as the positive control. Untreatedcontrols are indicated by triangles. Values are mean±SE of resultsobtained from three independent experiments. **P<0.01 compared tountreated control.

FIG. 5. Proliferation of human corneal epithelial cells (HCEC) inresponse to CAP37 using the CyQuant assay. CAP37 significantly(***P<0.001) affects the proliferation of HCEC in a time and dosedependent fashion. Levels of proliferation obtained with 1000 and 2000ng/ml of CAP37 were comparable to those obtained with the two positivecontrols epidermal growth factor (EGF) and hepatocyte growth factor(HGF). Data are ±SE of 4 independent experiments performed intriplicate.

FIG. 6. Migration of HCEC in response to CAP37 using the Boydenchemotaxis chamber assay. CAP37 is maximally chemotactic in the range of500-1000 ng/ml and was reduced but still measurable at 2000 ng/ml. Thelevels of migration were comparable to those obtained with the positivecontrol, platelet derived growth factor (PDGF). Data are mean± of 3independent experiments performed in triplicate. *P<0.05 and **P<0.01compared to the untreated buffer control.

FIG. 7. Inhibition of HCEC migration in response to CAP37 using aspecific antiserum to CAP37. Dose response inhibition of the chemotacticresponse, with significant inhibition (**P: <0.01) obtained with theantibody at 1:10 dilution. No inhibition obtained on the chemotacticactivity of PDGF for HCEC.

FIG. 8. RT-PCR analysis of HCEC for adhesion molecules ICAM-1, VCAM-1,PECAM-1 and E-selectin. ICAM-1 is constitutively expressed and wassignificantly upregulated in the presence of CAP37. PECAM-1 was alsoupregulated by CAP37. There was no upregulation of VCAM-1 and E-selectinmRNA expression in response to CAP37 treatment. +Ve=positiveTNFα-treated control; Unt=untreated; CAP37=CAP37 treated for 1, 2, 4, 6,12, and 24 hr.

FIG. 9. Kinetic expression of PECAM-1 in response to CAP37 measured byflow cytometry. Grey shaded area=isotype control, light line=untreatedcontrol, dashed line=TNF-α control, dark line=CAP37 at 1 μg/ml.Upregulation is significant between 6 and 12 hr.

FIG. 10. Kinetic expression of CD49c integrin molecule in response toCAP37 measured by flow cytometry. Initial upregulation of CD49c at 4 hrwith sustained protein expression through 24 hr. Grey shadedarea=isotype control, light line=untreated control, dark solidline=CAP37 treatment.

FIG. 11. Kinetic expression of CD29 integrin molecule in response toCAP37 as measured by flow cytometry. Grey shaded area=isotype control,light line=untreated control, dashed line=TNF-α control, dark solid line═CAP37 treatment.

DETAILED DESCRIPTION OF THE INVENTION

CAP37 (Cationic Antimicrobial Protein of M_(r) 37 kDa) is aninflammatory mediator which plays an important role in host defense andinflammation in the systemic circulation (11-15). PMN-CAP37 (SEQ IDNO:1) is constitutively expressed in the granules of humanpolymorphonuclear neutrophils (PMNs) and in the α granules of platelets(16-17), and due to its strong antibiotic activity was viewed as part ofthe oxygen-independent killing mechanism of the PMNs (18-20). The nativeprotein (PMN-CAP37) is particularly potent against the Gram negativebacteria including Escherichia coli, Salmonella typhimurium andPseudomonas aeruginosa (18-20). Peptides based on the native CAP37sequence have demonstrated antibiotic activity against the Gram positivebacteria, Enterococcus faecalis and Staphylococcus aureus (11). Inaddition to its effects on bacteria, CAP37 has many important functionaleffects on mammalian cells. CAP37 exerts powerful chemotactic activityfor monocytes (13) and regulates endothelial cell functions, such asstimulating protein kinase C (12).

CAP37 proteins and peptides derived therefrom can be used as atopical/oral/intravenous/intravitreal antibiotic for the treatment ofocular bacterial infections in mammals including humans, primates,rabbits, livestock animals and ungulates, for example. CAP37 and CAP37peptides described herein can also be used to promote healing of cornealwounds and ulcers that may not have an infective component, such asthose due to injury by foreign objects or trauma. CAP37 and CAP37peptides described herein can also be used to treat contact lenses, tosterilize the lenses and inhibit infections caused by bacteria on thelenses. Mammalian corneal transplants can also be stored in mediacontaining CAP37 and/or CAP37 peptides as described herein.

The present invention contemplates these treatments using a CAP37protein (native, synthetic, or recombinant) such as a CAP37 shown in SEQID NO:1, or SEQ ID NO:2. The present invention also contemplates the useof CAP37 peptides including CAP37 peptide 20-44 (SEQ ID NO: 3), CAP37peptide 23-42 (SEQ ID NO:4), CAP37 peptide 102-122 (SEQ ID NO:5), CAP37peptide 120-146 (SEQ ID NO:6), and monocysteine derivatives of CAP37peptide 23-42 and CAP37 peptide 20-44, (including peptides of SEQ IDNO:7 and SEQ ID NO:8) having the formula, for example:

R—H—X₁—X₂—X₃—X₄—X₅—X₆—X₇—H—X₈—R—X₉—X₁₀-M-X₁₁—X₁₂—X₁₃—X₁₄—X₁₅

wherein X₁ and X₉ are phenylalanine and/or tyrosine; X₂ and X₁₅ arecysteine, serine, and/or threonine; X₃ and X₄ are glycine and/oralanine; X₅-X₈, X₁₀, X₁₂ and X₁₃ are alanine, leucine, isoleucine and/orvaline; X₁₁ is serine and/or threonine; X₁₄ is serine, threonine,histidine, arginine or lysine; R is arginine; H is histidine; M ismethionine; and with the proviso that one of X₂ and X₁₅ is cysteine andone of X₂ and X₁₅ is serine or threonine.

To investigate the biological significance of CAP37 in cornealinfection, inflammation and healing, we used a well characterized invivo rabbit model of S. aureus keratitis (21, 22). An unexpected andsurprising observation was the expression of a CAP37 protein in cornealepithelial cells, stromal keratocytes, ciliary epithelium, relatedlimbus and ciliary vascular endothelium and bulbar conjunctiva.Particularly striking was the extremely strong staining for CAP37 incorneal epithelium (23). The in vivo studies outlined here demonstratethe kinetics of expression of CAP37 in extra-neutrophilic sitesincluding corneal epithelium and stromal keratocytes. These findingswere further dissected using in vitro studies in which human cornealepithelial cells and stromal keratocytes were used to determine themechanism of induction of CAP37 in these cells. Molecular cloning ofcorneal epithelial-derived CAP37 (EPI-CAP37-SEQ ID NO:2) was undertakento confirm our immunocytochemical analysis that the cornealepithelial-derived protein was unequivocally CAP37. The results of thepresent work indicate that CAP37 has far wider ranging effects on theinflammatory process than acting solely as an antibiotic and plays asignificant role in the sequence of events involved in leukocyteemigration and epithelial-leukocyte interactions in the inflamed corneafollowing infection.

Corneal epithelial wound healing has been described as comprising threesequential events: cell migration, cell proliferation and cell adhesion(24-26). We addressed the effect of CAP37 in vitro on these threecritical elements of wound healing. Corneal epithelial cellproliferation was assessed using the CyQuant proliferation assay. Cellmigration was determined by measuring chemotaxis using the modifiedBoyden chemotaxis chamber assay. Migration of leukocytes from thevasculature is dependent on the upregulation of adhesion molecules,therefore we measured the effect of CAP37 on upregulation of E-selectin,intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesionmolecule-1 (VCAM-1), and platelet endothelial cell adhesion molecule-1(PECAM-1) on human corneal epithelial cells (HCEC). Since attachment ofthe newly formed epithelium to the extracellular matrix is essential forcompleting the healing process we measured adhesion molecules such asβ1, β2, β3, β4, αv, α1, α2, α3 and α4 that are capable of binding tofibronectin, laminin and other extracellular matrix proteins (27)contributing to the formation of attachments and adhesion, therebyaiding the healing process.

Corneal Expression of CAP37

Methods

In Vivo Model of Staphylococcus aureus Keratitis

A rabbit model of S. aureus keratitis was used to determine thelocalization of CAP37 in the eye in response to infection. The model iswell established and the methodology published previously (21-22).Maintenance of animals and all in vivo experimentation was performedaccording to institutional guidelines and the Association of Research inVision and Opthalmology resolution on the use of animals in research asdetailed (http://www.arvo.org/aboutarvo/animalst.asp). Briefly, NewZealand white rabbits (2.0-3.0 kg) were injected intrastromally withapproximately 100 cfu of log phase S. aureus (RN6390 a wild-type straingenerously provided by Dr. Ambrose Cheung, Rockefeller University, NewYork, N.Y.) (28). The contralateral eye was injected intrastromally witheither phosphate buffered saline (PBS, 0.01 M, pH 7.4 containing 0.15 MNaCl; sham control) or was left undisturbed (absolute control). Therabbit eyes were assessed every 5 hr post infection by slit lampexamination (TOPCON BIOMICROSCOPE SL-5D, Kogaku Kikai K.K., Tokyo,Japan) (21, 22, 29). The course and severity of S. aureus keratitiscaused by strain RN6390 in these experiments was found to be similar toour previous reports (21, 22, 29). Eyes were enucleated at 5, 10, 15, 20and 25 hr post infection and processed for histologic analysis by fixingin 10% formalin for 24 hours. Tissue embedding, processing andsectioning was performed according to standard histologic techniques(Dean McGee Eye Institute, Histology Service Facility, Oklahoma City,Okla.).

Cell Culture

Immortalized human corneal epithelial cells (HCEC) provided by Dr.Araki-Sasaki, Suita, Japan were maintained as previously published (30).Briefly, HCEC were cultured in defined keratinocyte serum-free medium(GIBCO BRL, Grand Island, N.Y.) containing 1% penicillin-streptomycin(GIBCO BRL). Human stromal keratocytes were derived from donor corneas(North Florida Lions Eye Bank, Jacksonville Fla.) and cultured inDulbecco's modified Eagle's medium (DMEM, Mediatech, Herndon, Va.)containing 10% fetal bovine serum (FBS, HYCLONE LABORATORIES, Logan,Utah) and 1% penicillin-streptomycin (GIBCO BRL) according to ourprevious methods (31). Media changes were made every two to three days,and cells were subcultured (0.25% trypsin-1 mM EDTA at 37° C. for 5minutes, GIBCO BRL) when they reached 70% confluence at a split ratio of1:3. For measurement of cell adhesion molecules, the cells were detachedusing 5 mM EDTA alone (37° C. for 10 min). Cells were transferred toserum free basic medium overnight before the start of each experiment.

Recombinant CAP37

Functionally active recombinant CAP37 (rCAP37) was produced using aRSV-PL4 expression system in human 293 cells (32). The recombinantprotein was characterized as to amino acid sequence, SDS polyacrylamidegel electrophoresis and western blots and was shown to be identical tonative PMN-derived CAP37. All preparations of rCAP37 comprised <0.1endotoxin units/μg as determined by the limulus amebocyte lysate assay(QCL 100, WHITTAKER BIOPRODUCTS, Walkersville, Md.) performed exactlyaccording to the manufacturer's instructions.

Immunohistochemistry

The immunohistochemical analysis performed on paraffin-embeddedformalin-fixed rabbit eyes was according to previously published methods(13, 33). We used a previously characterized monospecific mouseanti-CAP37 antiserum (13) and the Vectastain™ avidin-biotin-complex(ABC) Elite technique (VECTOR LABORATORIES, Burlingame, Calif.) todetect CAP37. Briefly, 5 μm sections were cut along the optical axis andfollowing the various blocking steps (33) were incubated in the primaryantibody (mouse anti-human CAP37 at 1:1000 dilution in PBS containing0.1% normal goat serum and 0.1% bovine serum albumin) for 60 min at roomtemperature. Following three washes in buffer the slides were incubatedfor 30 minutes in the secondary antibody (biotinylated goat anti-mouseIgG, VECTASTAIN ABC Elite, VECTOR LABORATORIES) and then processedexactly as described in our previous publication (33). In order todetermine non-specific staining, negative controls without the primaryantibody, normal mouse serum, and immunoadsorbed anti-CAP37 antiserumwere incorporated in each experiment. Tissues were viewed under anOlympus BH-2 (Hitschfel Instruments, Inc, Lake Success, N.Y.) microscopeand photographs taken using an Olympus C-35AD4 camera.

For immunocytochemical analysis of HCEC and stromal keratocytes inculture, the cells were cultured on coverslips (CORNING COSTAR, Acton,Mass.) placed within 24-well tissue culture plates (CORNING COSTAR)until they reached 70% confluence and immunostained for CAP37 asdescribed above except for the following changes. Cells were fixed informol-acetone, pH 7.4 for 60 s at 4° C. (13) and were stained using themouse anti-human CAP37 antiserum (1:500 dilution).

In Vitro Induction of CAP37 in HCEC and Keratocytes

To determine if pro-inflammatory cytokines could induce CAP37 in HCECand keratocytes we treated these cell cultures with TNF-α (0-10 ng/ml,Boehringer-Mannheim, Indianapolis, Ind.) and IL-1β (0-20 ng/ml, ENDOGEN,Woburn, Mass.) for 0-24 hr and assayed the cells immunocytochemicallyfor the presence of CAP37 protein as described above. Untreated cellcultures were included for each test sample. In addition to proteindetection, upregulation of CAP37 mRNA in response to TNF-α and IL-1β wasmeasured by RT-PCR as described below.

RT-PCR

Cultured HCEC were treated with 5 ng/ml TNF-α and 10 ng/ml IL-1β for 0-8hr at 37° C. Total cellular RNA was isolated from untreated and treatedHCEC according to vendor specifications (TRIzol™, Gibco BRL). Afterreverse-transcription of 5 μg of total RNA by random oligonucleotidepriming (hexanucleotide mix, BOEHRINGER-MANNHEIM, GmbH, Germany), theresulting single stranded cDNA was amplified by PCR (PERKIN ELMER 2400thermocycler, Norwalk, Conn.) using CAP37 specific primers(CAGAATCAAGGCAGGCACTTCTGC (SEQ ID NO:9) and GAGAACACCATCGATCGAGTCTCG(SEQ ID NO:10)) designed for a 597 bp internal fragment of HL60-CAP37(34). The reaction conditions for reverse transcription were 80 units ofRNAse inhibitor (SIGMA), 8 μl of 5× strand buffer, 2 μl of randomhexanucleotide mix, 1 mM dNTPs (GIBCO BRL), 10 mM DTT (GIBCO BRL), and400 units of M-MLV RT (GIBCO BRL) in a total volume of 100 μl. Thereaction mix was incubated at 37° C. for 50 min followed by incubationat 70° C. for 15 min. The PCR mix (1.5 mM MgCl₂, 0.2 mM dNTPs, 1.26 μMof each primer and 1 unit Taq polymerase, GIBCO BRL) was amplified for30 cycles. Amplified DNA fragments were separated by electrophoresis ona 1% agarose gel and visualized by exposure to UV after ethidium bromide(0.5 μg/ml) staining. To assess the integrity of the cDNA, primers forhuman β-actin were used.

Molecular Cloning and Sequencing of HCEC CAP37

The cDNA products from the above RT-PCR were excised from the agarosegel and purified with the GENE CLEAN II KIT (BIO 101, Vista Calif.) andthen cloned using the TA CLONING KIT (INVITROGEN, Carlsbad, Calif.)according to the manufacturer's instructions. Ten white transformantsfrom each treatment were chosen for plasmid DNA isolation andpurification (WIZARD PLUS SV miniprep DNA purification system, Promega,Madison Wis.). Plasmids were sequenced in both forward and reversedirections using the T7 and M13 reverse primers from 6 different clonesfrom three independent clonings. The resulting sequences were alignedusing Pôle Bio-Informatique Lyonnais, Network Protein sequence @nalysis(35) for DNA and the consensus sequence compared against the HL-60 CAP37cDNA sequence (34).

Flow Cytometry

Flow cytometry was used to assess the upregulation of ICAM-1 and VCAM-1on HCEC in response to CAP37 treatment. HCEC were cultured as describedabove and treated with CAP37 (0-2000 ng/ml) for 0, 2, 6, 24, 48 and 72hr. A corresponding culture was left untreated at each time point.Following treatment with CAP37, cells were detached with 5 mM EDTA (pH7.4, Fisher Scientific), washed twice in PBS and fixed with 0.125%paraformaldehyde (J.T. Baker, Phillipsburg, N.J.) overnight at 4° C. Thecells were washed in PBS and then incubated in 0.5% normal goat serumand 0.5% BSA in PBS for 30 min to block non-specific binding sites. Fordetermination of ICAM-1 expression, cells were incubated in the primaryantibody (FITC-labeled mouse anti-human ICAM-1, BIOSOURCE, Camarillo,Calif.) at 10⁶ cells/10 μl at 4° C. for 1 hr. Cells were washed in PBSand analyzed by flow cytometry (FACSTAR, BECTON DICKINSON, San Jose,Calif.). For detection of VCAM-1 expression, cells were incubated withunlabeled primary antibody (monoclonal mouse anti human VCAM-1, ENDOGEN,Woburn, Mass. at 2 μg/10⁶ cells) followed by FITC-labeled goatanti-mouse IgG (PHARMINGEN, San Diego, Calif.) at 5 μg/10⁶ cells andincubated at 4° C. for 30 min. The isotype control for these studies wasFITC-labeled mouse isotype IgG₁ (PHARMINGEN). The positive control usedin these studies was TNF-α (5 ng/ml). At least ten thousand cells wereanalyzed for each sample.

Statistical Analysis

Data from the adhesion molecule studies are presented as mean±SE. Groupswere compared by unpaired student's t-test followed by ANOVA. P<0.05 wasconsidered significant.

Results

In Vivo Expression of CAP37 in S. aureus Keratitis Model

Immunohistochemical analysis was performed on tissue sections obtainedfrom eyes at 5, 10, 15, 20 and 25 hr post injection of S. aureus. Theinitial detection of CAP37 was made in the limbal epithelium and to alesser extent in the corneal epithelium at 5 hr (FIG. 1 a). Staining forCAP37 was not observed in sham-injected eyes at 5 hr post infection(FIG. 1 b) or at the later time points (not shown). By 10 hr postinfection, strong staining for CAP37 was demonstrated in the cornealepithelium (FIG. 1 c), ciliary epithelium, related limbus and ciliaryvascular endothelium (FIG. 1 d), and bulbar conjunctiva in rabbitsinjected with S. aureus. Staining for CAP37 was not observed in sectionsstained with normal mouse serum (FIG. 1 e) or with antiserumimmunoadsorbed with CAP37 (FIG. 1 f). The antibody control in FIGS. 1 eand 1 f indicate the specificity of the reaction for CAP37. No PMN orother leukocytic infiltration was observed in the cornea at the 10 hrtime point. However, a few PMN were seen in the bulbar conjunctiva andthe corneal limbus. The strong staining for CAP37 in the cornealepithelium persisted up to 15 hr (FIG. 1 g) and began to wane by 20 hr(FIG. 1 h). Staining for CAP37 in stromal keratocytes was more marked atthe 15 hr time point than at the 10 hr time point. An importantobservation in this in vivo model was that CAP37 induction in vivo wasobserved before leukocyte infiltration, which in our studies occurred at15 hr post infection (FIG. 1 g). Neutrophils were first seen in thestroma at approximately 15 hr post injection of the pathogen, and thenbegan to accumulate at the base of the epithelial layer between 20 and25 hr post infection (FIG. 1 h). Obvious stromal edema and severeanterior chamber inflammatory reaction were also readily observed at thelater time points. With time, the inflammatory reaction became moresevere; clumps of bacteria were evident within the stroma but the levelsof CAP37 in the corneal epithelium and stromal keratocytes diminished.It is important to note that PMN continued to stain for CAP37 throughoutall the time points (FIG. 1 h), even though epithelial CAP37 was reducedor could no longer be detected.

In Vitro Expression of CAP37 in Human Corneal Epithelial Cells andKeratocytes

Since CAP37 was detected in the corneal epithelium and stromalkeratocytes in vivo in response to the intrastromal Gram-positiveinfection but was not present in normal, uninfected eyes, thepossibility that CAP37 was induced in response to inflammatory mediatorsand/or cytokines generated as part of the host's defense response to theinfection was studied. Two proinflammatory cytokines, TNF-α and IL-1β,are known to be present during the acute stages of a wide range ofinflammatory situations (36-39), and have been implicated in geneexpression of other chemoattractants such as IL-8 (40-41). Usingimmunocytochemistry and RT-PCR we explored the possibility that theymight regulate CAP37 expression in HCEC and keratocytes. Theimmunocytochemical data presented in FIG. 2 demonstrate that CAP37protein is induced in HCEC in response to TNF-α (FIG. 2 a) and IL-1β(FIG. 2 b). Detection of CAP37 protein was observed as early as 60 minin the TNF-α treated cells and appeared maximum at 24 hr. Expression ofCAP37 in response to IL-1β was observed at a later time point (4 hr) andlike TNF-α appeared to have its maximum effect at 24 hr. There was noconstitutive expression of CAP37 protein in untreated HCEC (FIG. 2 c).Antibody controls using immunoadsorbed anti-CAP37 antiserum showed nostaining, indicating the specificity of this reaction (FIG. 2 d).Stromal keratocytes treated with TNF-α (FIG. 2 e) and IL-1 (not shown)showed the induction of CAP37 protein. Once again there was noconstitutive expression of CAP37 in these cells as indicated by a lackof staining with the anti-CAP37 antiserum in the untreated cell cultures(not shown). The specificity of this reaction was demonstrated by thelack of staining with the immunoabsorbed antibody control (FIG. 2 f).

We corroborated the immunocytochemical data above using RT-PCR. Humancorneal epithelial cells treated with TNF-α (FIG. 3 a) and IL-1β (FIG. 3b) showed a time-dependent expression of CAP37 mRNA. Untreated HCEC donot express CAP37 mRNA. However, on treatment with the proinflammatorycytokine, TNF-α, HCEC express CAP37 mRNA as early as 15 minutes. Theselevels are maximum between 30 min and 2 hr, and reduced by 4 hr. IL-1also induced CAP37 mRNA in HCEC. However as demonstrated in FIG. 3 b,the initial expression of CAP37 mRNA is delayed and is not detecteduntil 1 hr post stimulation. Furthermore, the effect is more sustainedthan with TNF-α, as the message can be detected even at 6 hr. Thesefindings corroborate our immunocytochemical data in which TNF-α inducedprotein at an earlier time point and that the more intense staining ofCAP37 was obtained in response to IL-1β.

Molecular Cloning of Human Corneal Epithelial Cell CAP37 (EPI-CAP37)

To determine whether EPI-CAP37 (SEQ ID NO:2) was similar to PMN-CAP37(SEQ ID NO:1) we undertook the cloning of HCEC-CAP37. Total cellular RNAwas isolated from HCEC treated with TNF-α for 2 hr and cDNA synthesisperformed according to the methodology described above. RT-PCR was usedto amplify the CAP37 gene from HCEC using the pair of oligonucleotideprimers as described in the methods and based on a previously publishedcDNA sequence of CAP37 (34). EPI-CAP37 has the same sequence as residues20-218 of PMN-CAP37 (SEQ ID NO:1) except for amino acid residue atposition 113 of EPI-CAP37 (SEQ ID NO:2), wherein a histidine residueconsistently replaced the arginine residue found at the correspondingposition in PMN-CAP37 (i.e., residue 132 of SEQ ID NO:1).

Upregulation of ICAM-1 on Cultured HCEC

In vitro studies were undertaken to investigate the effect of CAP37 onthe upregulation of ICAM-1, on HCEC. Cells were treated with CAP37(0-2000 ng/ml) for 0-72 hr and levels of ICAM-1 measured using flowcytometry. ICAM-1 was upregulated by CAP37 in a dose-dependent fashion,with maximum upregulation obtained with 1000-2000 ng/ml of CAP37 (FIG. 4a). These levels were comparable to those obtained with the positivecontrol TNF-α (5 ng/ml). Lower, yet significant levels of ICAM-1 wereobtained with CAP37 at concentrations between 10 and 500 ng/ml. Kineticstudies (FIG. 4 b) indicated that HCEC did not constitutively expressICAM-1 and that no upregulation of ICAM-1 could be detected by flowcytometry at the early time point of 2 hr. However by 6 hr, significantupregulation of ICAM-1 was observed. The levels declined by 24 hr, butwere still above the untreated levels.

As noted previously, extravasation of leukocytes from the circulationinto tissue sites is an integral feature of the host response to injuryand inflammation (42). By virtue of their ability to engulf and destroybacteria, eliminate toxins and secrete numerous soluble mediators,leukocytes are capable of restricting and limiting the spread ofinfection. In the acute stages of most infections, the predominant celltype is the PMN (42, 43). This observation held true in our in vivorabbit model of S. aureus keratitis, where the primary leukocyteobserved in the initial 25 hr period following infection was the PMN.The rabbit bacterial keratitis model indicated the expected expressionof CAP37 in the granules of migrating PMN. However, a surprising andunexpected observation was the expression of CAP37 in corneal epithelialcells, stromal keratocytes, ciliary epithelium, related limbus andciliary vascular endothelium and bulbar conjunctiva. Particularlystriking was the extremely strong staining for CAP37 in cornealepithelium at 10 hr post infection. The induction of CAP37 in the corneaoccurred prior to the emigration of PMN, which in this model occurredapproximately 15 hr post infection. The path of migration of PMNappeared to be from ciliary and limbal vessels through the stroma to thebasal aspects of the epithelial layer, where large numbers of PMN wereseen to accumulate.

Clearly, as indicated by the present results, extra-neutrophilic CAP37is induced in response to infection or an inflammatory stimulus, sincesham-injected animals do not show staining for CAP37. These aresignificant findings, since the extra-neutrophilic localization of CAP37in ocular tissue in response to infection has not been reportedpreviously. Our data indicate that the source of CAP37 in the cornealepithelium is endogenous during the early stages of infection. This isbased on our unequivocal observations that corneal CAP37 is seen in theabsence of and prior to PMN extravasation. Thus the staining observed inthe epithelium could not be due to exogenously released CAP37 from PMN.Our in vitro studies depicted in FIGS. 2 and 3 support the concept thatCAP37 can be induced in any ocular infection in which TNF-α and IL-1βare generated.

Our in vitro studies show that the pro-inflammatory mediators TNF-α andIL-1β regulate CAP37 expression in corneal epithelial cells and stromalkeratocytes in a time- and dose-dependent fashion. Untreated cells didnot display CAP37 message or protein, indicating that it is notconstitutively expressed in either of these cells. This is the firstdemonstration of the expression of a monocyte chemoattractant in HCEC inresponse to cytokines. The induction of monocyte chemotactic protein-1(MCP-1), RANTES (44), and GROα (45), members of the C—C chemokine familywith chemotactic effects on monocytes has been demonstrated in stromalkeratocytes but not in HCEC. On the other hand, expression of C—X—Cchemoattractants such as IL-8 with potent effect on PMN migration can beinduced in HCEC (40) and stromal keratocytes (31). These studiesdemonstrate a novel localization of the inflammatory mediator CAP37 andindicate that these new properties contribute to its role in hostdefense in ocular inflammation.

Modulation of Corneal Epithelial Cell Functions by CAP37

Methods

Cell Culture

Immortalized human corneal epithelial cells (HCECs,) provided by K.Araki-Sasaki, (Suita, Japan) (30) were grown and maintained in definedkeratinocyte-serum free media (GIBCO BRL, Grand Island, N.Y.) containing1% penicillin-streptomycin (GIBCO BRL) as described previously (31).Media changes were made every two to three days and cells weresubcultured (0.25% trypsin-1 mM EDTA at 37° C. for 5 minutes, GIBCO BRL)when they reached 70% confluence at a split ratio of 1:3.

Recombinant CAP37

Functionally active recombinant CAP37 (rCAP37) was produced andcharacterized as described above.

Cell Proliferation

Human corneal epithelial cells were seeded onto 48 well tissue cultureplates (7.5×10³ cells/well, FALCON, Franklin Lakes, N.J.) and culturedas described above.

Cultures were changed to growth factor-free basic medium overnight andtreated with various concentrations of CAP37 (0-2000 ng/ml) for 48-72hrs. Recombinant human Epidermal Growth Factor (EGF 50 ng/ml, BECTONDICKINSON, Bedford, Mass.) and recombinant human Hepatocyte GrowthFactor/Scatter Factor (HGF/SF 20 ng/ml, BECTON DICKINSON) were used aspositive controls and growth factor-free basic medium as negativecontrol. The medium was aspirated and new medium with CAP37 or growthfactors were added to the cultures every 24 hr. The CyQUANT CellProliferation Assay Kit (MOLECULAR PROBES, Eugene, Oreg.) was used toquantify cell proliferation exactly according to the manufacturer'sspecifications. Briefly, cells were frozen, thawed, and lysed with theaddition of the lysis buffer containing the green fluorescent dye,CyQUANT GR which binds to nucleic acids and the fluorescence levels readon fluorescent micro plate reader (fmax MOLECULAR DEVICES, Sunnyvale,Calif.) with filters for 485 nm excitation and 538 nm emission.

Chemotaxis Assay

Human corneal epithelial cells were cultured in basic medium overnight,detached using trypsin-EDTA as described above and resuspended at afinal concentration of 8×10⁵ cells/ml. Chemotaxis assays were performedusing the modified Boyden chamber assay described previously (13).Briefly, 200 (μl of cell suspension was added to the upper chamber andchemoattractants including recombinant CAP37 (10-2000 ng/ml) and thepositive control recombinant human Platelet Derived Growth Factor-BB(PDGF-BB, 10 ng/ml, Collaborative Biomedical Products, Bedford Mass.) in0.1% BSA (endotoxin-low-Sigma, St. Louis) in Geys' Buffer (GIBCO) wereadded to the lower chamber. The chambers were separated by an 8.0 μmpore membrane (13 mm polyvinylpyrrolidone-free, Whatman, Clifton, N.J.).Membranes were pre-coated with 50 μg/ml collagen type I rat tail(Collaborative Biomedical Products) in 0.02N acetic acid at roomtemperature for 1 hr and then air dried. Membranes were re-hydrated inbasic cell culture medium immediately prior to commencement of eachexperiment. The negative control in these experiments was 0.1% BSA inGeys' buffer. The chambers were incubated in a humidified atmosphere(37° C., 5% CO₂) for 4 hr, the filters were removed, stained withDIFF-QUICK (Dade Behring, Düdingen, Switzerland) and mounted withPermount (FISHER SCIENTIFIC, Pittsburgh, Pa.). The filters were viewedunder oil immersion (×400 magnification, BH-2, Olympus, Lake Success,N.Y.) and the total numbers of cells migrated through to the undersideof the filter were counted in five different fields on each slide.Triplicates were set up for each experimental point.

To assess whether CAP37 had chemokinetic properties, variousconcentrations of CAP37 (0, 10, 100 and 1000 ng/ml) were added to theupper chamber as well as to the lower chamber (0, 10, 100, 500, 1000ng/ml) and a checkerboard assay performed according to the methodologyof Zigmond and Hirsch (46).

To determine the specific interaction of CAP37 with HCEC, we used apreviously characterized polyvalent, monospecific rabbit antiserum toCAP37 (12) to inhibit the chemotactic activity of CAP37. CAP37 wasincubated with heat inactivated (56° C. for 30 min) rabbit antiserum atconcentrations of 1:10, 1:50, and 1:100 and chemotaxis assays performedas outlined above using 500 ng/ml (1.3×10⁻⁸ M)_(r)CAP37. Controlsincluded heat-inactivated antiserum alone, CAP37 alone, PDGF alone andPDGF plus antiserum.

Flow Cytometry

Flow cytometry was used to assess the upregulation of PECAM-1 (CD31),and the integrin molecules β1 (CD29), β2 (CD18), β3 (CD61), β4 (CD104),α1 (CD49a), α2 (CD49b), α3 (CD49c), α4 (CD 49d), and αv (CD51). Humancorneal epithelial cells were cultured as above and treated with CAP37(0-2000 ng/ml) for 0-72 hr. A corresponding culture was left untreatedat each time point. Following treatment with CAP37, cells were detachedwith 0.25% trypsin in 1 mM EDTA (pH 7.4, FISHER SCIENTIFIC, Pittsburgh,Pa.), washed twice in PBS and fixed with 0.125% paraformaldehyde (J.T.BAKER, Phillipsburg, N.J.) overnight at 4° C. The cells were washed inPBS and then incubated in 0.5% normal goat serum and 0.5% bovine serumalbumin (BSA) in PBS for 30 min to block non-specific binding sites.Cells were incubated in the primary antibody (at concentrationsdescribed below) at 4° C. for 1 hr followed by the secondary antibody(FITC-goat anti-mouse IgG, PHARMINGEN, San Diego, Calif.) at 0.5 μg/10⁶cells and incubated at 4° C. for 30 min. The isotype control for thesestudies was FITC-labeled mouse isotype IgG₁ (PHARMINGEN). The cells wereanalyzed by flow cytometry (FACS Calibur, BECTON DICKINSON, San Jose,Calif.). At least ten thousand cells were analyzed for each sample.

Antibodies

The primary antibodies and the concentrations used in the flow cytometryexperiments are as follows: mouse anti-human PECAM-1 (CD31) monoclonalantibody clone HEC7 (0.5 μg/10⁶ cells, ENDOGEN, Woburn, Mass.), mouseanti-human very late antigen 1α (VLA-1α, or CD49a) monoclonal antibodyclone SR84 (0.5 μg/10⁶ cells, PHARMINGEN), mouse anti-human VLA-α₂(CD49b) monoclonal antibody clone AK-7 (0.125 μg/10⁶ cells, PHARMINGEN),mouse anti-human α3 (CD49c) monoclonal antibody clone C3II.1 (0.125μg/10⁶ cells, PHARMINGEN), mouse anti-human VLA-4 (α4) monoclonalantibody clone 2B4 (1 μg/10⁶ cells, R & D systems, Minneapolis, Minn.),mouse anti human α5 (CD49e) monoclonal antibody clone VC5 (0.125 μg/10⁶cells, PHARMINGEN), mouse anti-human β₁ (CD29) monoclonal antibody MAR4(2 μg/10⁶ cells, PHARMINGEN), mouse anti human β₂ integrin (CD18)monoclonal antibody clone 6.7 (0.5 μg/10⁶ cells, PHARMINGEN), mouse antihuman α_(v)β₃ (CD51/CD61) monoclonal antibody clone 23C6 (0.5 μg/10⁶cells, PHARMINGEN), and mouse anti human integrin β₄ (CD104) monoclonalantibody clone 450-11A (1.0 μg/10⁶ cells, PHARMINGEN). A purified mouseIgG₁ κ monoclonal immunoglobulin isotype standard (clone MOPC-31C) wasused as the isotype matched control in the flow cytometry experiments.

RT-PCR

Cultured HCEC were treated with CAP37 (1 μg/ml) for 0-24 hr at 37° C.Total cellular RNA was isolated from untreated and treated HCECaccording to vendor specifications (TRIzol™, GIBCO BRL). Afterreverse-transcription of 5 μg of total RNA by SuperScript™ II RT(GIBCOBRL) the resulting single stranded cDNA was amplified by PCR(BIOMETRA TGRADIENT, Göttingen, Germany) using specific primers forICAM-1 ((GTCCCCCTCAAAAGTCATCC (SEQ ID NO:11) and AACCCCATTCAGCGTCACGT(SEQ ID NO:12)); VCAM-1 ((AGTGGTGGCCTCGTGAATGG (SEQ ID NO:13) andCTGTGTCTCCTGTCTCCGCT (SEQ ID NO:14)); PECAM-1 ((TTGCAGCACAATGTCCTCTC(SEQ ID NO:15) and AGCACAGTGGCAACTACACG (SEQ ID NO:16)); E-selectin((AGAAGAAGCTTGCCCTATGC (SEQ ID NO:17) and AGGCTGGAATAGGAGCACTCCA (SEQ IDNO:18)); and β-actin ((TACCTCATGAAGATCCTCA (SEQ ID NO:19) andTTCGTGGATGCCACAGGAC (SEQ ID NO:20))) synthesized by the MolecularBiology Resource Facility, University of Oklahoma Health SciencesCenter. The thermocycler conditions for ICAM-1 and VCAM-1 were 95° C.for 5 min initially, with 30 cycles at 95° C. for 1 min, 58° C. for 45sec, 72° C. for 1 min followed by a final extension at 72° C. for 7 min.The conditions for E-selectin were 95° C. for 5 min initially, with 30cycles at 94° C. for 1 min, 58° C. for 1 min, 72° C. for 1 min followedby a final extension at 72° C. for 5 min. The conditions for PECAM-1were 95° C. for 5 min initially, with 30 cycles at 95° C. for 45 sec,60° C. for 1 min, 72° C. for 1 min followed by a final extension at 72°C. for 5 min.

Amplified DNA fragments were separated by electrophoresis on a 1%agarose gel and visualized by exposure to UV after ethidium bromide (0.5μg/ml) staining. Expected sizes for ICAM-1, VCAM-1, PECAM-1, E-selectinand β-actin were 943 bp, 700 bp, 677 bp, 315 bp and 267 bp,respectively. To assess the integrity of the cDNA, primers for humanβ-actin were used.

Statistical Analysis

Data from proliferation and chemotaxis and adhesion molecule studies arepresented as mean±SE. Groups were compared by unpaired student's t-testfollowed by ANOVA. P<0.05 was considered significant.

Results

Proliferation of HCEC in Response to CAP37

CAP37 significantly affects the proliferation of HCEC (FIG. 5). Thisresponse is both dose- and time-dependent. At 48 hours post treatmentwith CAP37, there was a significant increase in proliferation over basallevels observed in culture medium alone. Levels of proliferationobtained with 1000-2000 ng/ml (2.7-5.4×10⁻⁸ M) of CAP37 were comparableto those obtained with the two positive controls, EGF and HGF. HCECcontinued to proliferate with time and an approximately two- tothree-fold increase in cell numbers was obtained at 72 hr post treatmentwith 1000 ng/ml and 2000 ng/ml of CAP37 respectively. The levelsobtained with EGF and HGF were similar to those obtained with 1000 ng/mlof CAP37.

Migration of HCEC in Response to CAP37

We investigated whether CAP37 was chemotactic for HCEC using themodified Boyden chemotaxis technique. Data shown in FIG. 6 indicate thatCAP37 is a strong chemoattractant for HCEC. It was maximally chemotacticin the range of 500 ng/ml to 1000 ng/ml and was reduced but stillmeasurably active at 2000 ng/ml. The levels of migration in response toCAP37 were comparable to those obtained with the positive control, PDGF.The dose response obtained with CAP37 shows the typical bell-shapedcurve indicative of a chemoattractant. However, an important issue thatrequires clarification when determining movement of cells in response toa mediator is whether this migration is due to directed movement(chemotaxis) as opposed to merely accelerated random motion(chemokinesis). The checkerboard assay (46) has been traditionallyemployed to distinguish chemotaxis from chemokinesis. Our experimentsdemonstrate that the effect of CAP37 on HCEC is predominantlychemotactic (Table I). Most chemoattractants display a certain level ofchemokinesis particularly at higher concentrations (46). The dataobtained clearly demonstrate that there is an increase in numbers ofcells migrating across the filter when increasing concentrations ofCAP37 are present in the lower chamber, but absent from the upperchamber i.e. standard chemotaxis assay (Table I, row 1). The addition ofCAP37 to the upper chamber resulted in a reduction of the chemotacticgradient across the membrane, with corresponding reduction in levels ofmigration. The values on the diagonal in Table I represent chambers thatwere set up with equal concentrations of CAP37 across the membrane andclearly indicate that the levels of migration are not significantlygreater than background. The values in Table I are represented as totalnumbers of cells migrated rather than percent of control to indicate theabsolute values of cells migrating to the underside of the filter.

TABLE I Concentration Number of cells migrated CAP37 above theConcentration of CAP37 below the filter (ng/ml) filter (ng/ml) 0 10 100500 1000 0 24.06 ± 2.80 28.56 ± 7.64 39.82 ± 6.20 68.89 ± 7.70 *** 67.34± 8.42 *** 10 ND 27.63 ± 3.03 39.80 ± 6.93 52.40 ± 15.76 *** 62.73 ±9.66 *** 100 ND ND 36.63 ± 6.48 ND 48.57 ± 15.58 *** 1000 ND ND 28.08 ±2.76 ND 38.13 ± 6.07 Determination of chemokinetic properties of CAP37by the checkerboard assay for HCEC. CAP37 has some chemokinetic activityat the higher concentrations, but it contributes little to the overallchemotactic effect on HCEC.

To demonstrate the specificity of this chemotactic response, an antibodypreviously shown to be specific for CAP37 was used to inhibit themigration of cells in response to CAP37. FIG. 7 indicates a doseresponse inhibition of the chemotactic response, with significantinhibition (p<0.01) obtained with the antibody at 1:10 dilution. Aspredicted, the antibody did not have an inhibitory effect on thechemotactic activity of PDGF for HCEC.

Effect of CAP37 on Adhesion Molecules on HCEC

RT-PCR was performed using primers specific for ICAM-1, VCAM-1, PECAM-1and E-selectin. Treatment of HCEC with CAP37 indicates a clearupregulation of ICAM-1 message beginning at 2 hr and lasting through 24hr (FIG. 8). Maximum expression of ICAM-1 message was seen between 2 and4 hr. PECAM-1 was also upregulated by CAP37. Unlike the upregulation ofICAM-1 message, upregulation of PECAM-1 message was transient. It wasdetected at 2 hr after stimulation, maximum at 4 hr and could notdetected after 6 hr. HCEC did not show increase in mRNA expression ofVCAM-1 and E-selectin in response to CAP37 treatment.

The expression of PECAM-1 in response to CAP37 treatment was furtherconfirmed using flow cytometry (FIG. 9). Significant protein expressionwas observed on HCEC at 6 hr, was maintained through 12 hr and waned by24 hr, corroborating our findings in FIG. 8. The kinetics of thisresponse to CAP37 appeared to follow that of TNF-α up to 12 hr.Thereafter the effect of TNF-α was more sustained, lasting until 24 hr(not shown).

Upregulation of α1, α2, α3, α4, αv and β1, β2, β3, β4 integrins inresponse to CAP37 was also assessed using flow cytometry. Table IIsummarizes the data obtained from these analyses. Of the 8 integrinmolecules analyzed only two showed significant upregulation. CD49c (α3)was initially upregulated at 4 hr, and the level of protein expressionwas sustained through 24 hr (FIG. 10). CD49c protein levels on HCEC at48 and 72 hr returned back to constitutive levels (not shown). There washigh constitutive expression of CD49c, as indicated by strong stainingon untreated HCEC. The other integrin molecule to be upregulated byCAP37 was CD29 (β1). The upregulation is clearly significant by 6 hr,increases to maximum levels between 12 and 48 hr, and although reducedat 72 hr is still significantly elevated above background constitutivelevels (FIG. 11). The flow cytometry analysis indicates a low level ofconstitutive expression of CD29 which remains constant throughout alltime points in this experiment. TNF-α was used as the positive controlin these experiments.

TABLE II Integrin molecule Effect of CAP37 α1 (CD49a; VLA-α1)Constitutive expression - no upregulation α2 (CD49b; VLA-α2)Constitutive expression - no upregulation α3 (CD49c) High constitutiveexpression - significant upregulation α4 (CD49d) Low constitutiveexpression - no upregulation β 1 (CD29) Constitutive expression -significant upregulation β 2 (CD18) No constitutive expression - noupregulation β 4 (CD104) Constitutive expression - no upregulation αv β3 (CD51/CD61) Low constitutive expression - no upregulation Effect ofCAP37 on integrin molecules on HCEC

As indicated by the results, the presence of the novel inflammatorymolecule CAP37 has been identified in the eye. The in vitro evidencepresented indicates its expression in HCEC and stromal keratocytes inresponse to inflammatory cytokines such as TNF-α and IL-1β. The resultsshow that CAP37 modulates corneal epithelial cell functions includingproliferation, migration and upregulation of adhesion moleculesimportant in epithelial-extracellular matrix interactions. In additionto upregulation of adhesion molecules important inepithelial-extracellular matrix interactions, CAP37 also regulates theexpression of adhesion molecules of the immunoglobulin superfamilyimportant in leukocyte-epithelial interactions. Specifically, CAP37upregulated the adhesion molecules ICAM-1 and PECAM-1. CAP37 modulatesinfections in the eye through its ability to act as an antibiotic,elicit leukocyte recruitment and affect corneal epithelial cellsfunctions, thereby regulating corneal inflammation and healing.

Utility

The present invention contemplates the use of a native, synthetic, orrecombinant CAP37, or peptide portions thereof, or derivatives thereof,as described herein, to treat various conditions of the eye includinginfections. The invention further contemplates the use of a native,synthetic, or recombinant CAP37, or peptide portions thereof, orderivatives thereof, in the treatment of corneal ulcers and wounds. Theinvention also contemplates the use of a native, synthetic, orrecombinant CAP37, or peptide portions or derivatives thereof, as adisinfectant for cleaning or sterilization of contact lenses and as astorage solution for preventing contact lenses from becomingcontaminated with bacteria while in contact lens storage cases. Theinvention also contemplates coating contact lenses with a native,synthetic, or recombinant CAP37, or an antibiotic peptide portions orderivatives thereof (and contact lenses thus coated), to inhibit,prevent or treat infections, bacterial keratitis and/or the growth ofbiofilms on or by contact lenses. The invention also contemplates amethod for storage of mammalian corneal tissue or transplants in mediacontaining a native, synthetic, or recombinant CAP37, or peptideportions or derivatives thereof, or at bactericidal concentrations foraseptic transportation and storage.

CAP37 peptides which can be used in the present invention are functional(antibiotic) and immunomodulatory peptides of CAP37 peptides of CAP37 orderivatives thereof and include, but are not limited to, peptide 20-44,peptide 23-42, peptide 102-122, peptide 120-146, and monocysteinederivatives of peptides 20-44 and 23-42 as described in U.S. Pat. No.6,107,460 which is hereby expressly incorporated by reference herein inits entirety and as referred to elsewhere herein.

More particularly the invention includes, but is not limited to:

-   1. Use of a native, synthetic, or recombinant CAP37, or peptides    thereof, and/or derivatives thereof, as described herein, as an    ocular antibiotic treatment, for conjunctivitis or bacterial    keratitis, particularly in those cases due to Pseudomonas aeruginosa    and Staphylococcus aureus.-   2. Use of a native, synthetic, or recombinant CAP37, or peptides    thereof, and/or derivatives thereof, as described herein, as a    cleaning and sterilization procedure for storing contact lenses in    storage cases. Since Pseudomonas aeruginosa is the most common    causative agent, contact lenses could be stored in a bactericidal    solution of a native, synthetic, or recombinant CAP37, or peptides    thereof, and/or derivatives thereof as described herein. This would    be an important mechanism to prevent or inhibit ocular infections    before they are initiated.-   3. Extended wear contact lenses could be manufactured with a surface    coating of a native, synthetic, or recombinant CAP37, or    bactericidal peptides thereof, and/or derivatives thereof, as    described herein, as a preventive method to prevent or inhibit    infections from occurring or biofilms from forming.-   4. Human corneal transplants could be stored in media containing    bactericidal quantities of a native, synthetic, or recombinant    CAP37, or peptides thereof, and/or derivatives thereof, as described    herein, during transportation and storage.-   5. A native, synthetic, or recombinant CAP37, or peptides thereof,    and/or derivatives thereof, as described herein, could be used in    treating ulcers and wounds of the cornea to promote healing.-   6. Use of a native, synthetic, or recombinant CAP37, or bactericidal    peptides thereof, and/or derivatives thereof, as described herein,    to treat serious bacterial infections which occur post-operatively.    For example, endophthalmitis, including post-operative    endophthalmitis due to coagulase negative Staphylococcus, is a major    problem. Infection of the conjunctival filtering bleb created by    glaucoma surgery, known as “blebitis”, due most commonly to    Staphylococcus aureus, Streptococcus and Hemophilus are further    targets for treatment with a native, synthetic, or recombinant    CAP37, or peptides thereof, and/or derivatives thereof, as described    herein.

The use of a native, synthetic, or recombinant CAP37, or peptidesthereof, and/or derivatives thereof, described herein, as antibiotics isadvantageous over other available therapies since its mode of action isdifferent from traditional antibiotics. Therefore the chances ofantibiotic resistant organisms arising as a result of this therapy arefar less than with traditional antibiotics. Since CAP37 is a naturallyoccurring protein or peptide, the chances of allergic reactions andtoxicity are less. It has activity with a relatively narrow spectrum,but is active against both Pseudomonas and Staphylococcus, the two mostcommon causative organisms of bacterial keratitis. Further, a native,synthetic, or recombinant CAP37, or peptides thereof, and/or derivativesthereof, are generally bactericidal rather than bacteriostatic.

A native, synthetic, or recombinant CAP37, or peptides thereof, and/orderivatives thereof, as described herein, are active against the twomost common causative organisms, but have limited activity against anumber of other Gram negative and Gram positive bacteria, therefore,treatment using them would not be overly toxic to normal flora. TheCAP37 peptides in particular are small, easily synthesized, and can bedelivered in required concentrations topically.

In one treatment protocol, the proteins or peptides described herein areprovided at a concentration of 200 μg/ml in a saline or “natural tears”solution, but may be at a concentration from about 10 μg/drop to 1000μg/drop (50 μl/drop). Drops may be administered to a subject's eye, forexample, every 15 minutes to 1 hour for the first 2-3 days of treatment,followed by dosing every 4 hours for 14 more days. The proteins orpeptides described herein could also be applied to the eye as anointment. The CAP37 proteins or peptides can be applied by intravitrealinjection for treatment of endophthalmitis in a manner well known tothose of ordinary skill in the art.

The following U.S. patents are hereby expressly incorporated herein byreference in their entirety: U.S. Pat. Nos. 5,607,916; 5,650,392;5,627,262; 5,877,151; 6,071,879; 6,107,460; 5,458,874; and 5,484,885.References cited herein are also expressly incorporated by referenceherein in their entireties.

All references, articles and patents cited herein are herebyincorporated herein in their entirety by reference.

While the invention is described herein in connection with certainembodiments so that aspects thereof may be more fully understood andappreciated, it is not intended that the invention be limited to theseparticular embodiments. On the contrary, it is intended that allalternatives, modifications and equivalents are included within thescope of the invention as defined by the appended claims. Thus theexamples described below, which include preferred embodiments, willserve to illustrate the practice of this invention, it being understoodthat the particulars shown are by way of example and for purposes ofillustrative discussion of preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description ofprocedures as well as of the principles and conceptual aspects of theinvention. Changes may be made in the formulation of the variouscompositions described herein or in the steps or the sequence of stepsof the methods described herein without departing from the spirit andscope of the invention as described and claimed herein.

REFERENCES

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1. A method of increasing proliferation and migration of cornealepithelial cells in the cornea of a mammalian subject having a cornealulcer or wound and increasing their adherence to the basement membraneof the cornea, comprising: providing a therapeutically-effective amountof a purified recombinant or synthetic CAP37 peptide comprising SEQ IDNO:5; and administering the therapeutically-effective amount of thepeptide to the eye of the mammal, wherein the peptide promotes healingof the corneal ulcer or wound by the increase in proliferation andmigration of corneal epithelial cells and by the increase in adherenceof the migrating corneal epithelial cells to the basement membrane ofthe cornea.
 2. The method of claim 1 wherein the mammal is a human.
 3. Amethod of increasing proliferation and migration of corneal epithelialcells in the cornea of a mammalian subject having a corneal ulcer orwound and increasing their adherence to the basement membrane of thecornea, comprising: providing a therapeutically-effective amount of apeptide consisting of SEQ ID NO:5; and administering thetherapeutically-effective amount of the peptide to the eye of themammal, wherein the peptide promotes healing of the corneal ulcer orwound by the increase in proliferation and migration of cornealepithelial cells and by the increase in adherence of the migratingcorneal epithelial cells to the basement membrane of the cornea.
 4. Themethod of claim 3 wherein the mammal is a human.